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Morphological changes in the retina of Aequidens pulcher (Cichlidae) after rearing in monochromatic light
Vision Research 39 (1999) 2441 – 2448
Morphological changes in the retina of Aequidens pulcher (Cichlidae) after rearing in monochromatic light R.H.H. Kro¨ger a,*, J.K. Bowmaker b, H.J. Wagner a a
Anatomisches Institut, Eberhard-Karls-Uni6ersita¨t Tu¨bingen, O8 sterbergstraße 3, 72074 Tu¨bingen, Germany b Institute of Ophthalmology, Uni6ersity College London, Bath Street, London EC1V 9EL, UK Received 8 May 1998; received in revised form 23 July 1998
Abstract We investigate the processing of chromatic information in the outer retina of a cichlid fish, Aequidens pulcher. The colour opponent response characteristics of some classes of cone-specific horizontal cells in the fish retina are the result of feedforward– feedback loops with cone photoreceptors. To interfere with the reciprocal transmissions of signals, animals were reared in monochromatic lights which preferentially stimulated the spectrally different cone types. Here we report the effects on the cones. Their absorbance spectra were largely unaffected, indicating no change in photopigment gene expression. Significant changes were observed in the cone outer segment lengths and the frequencies of spectral cone types. Quantum catch efficiency and survival of cones appear to be controlled in a spectrally selective way. Our results suggest that the retina responds to spectral deprivation in a compensatory fashion aimed at balancing the input from the different cone types to second order neurons. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Fish; Color vision; Photopigment gene expression; Photoreceptor survival; Adaptive plasticity
1. Introduction Investigations of chromatic information processing in the retina and its cellular substrate are critically dependent on unambiguous identification of spectral cone types (Stell, Barton, Ohtsuka & Hirano, 1994). Therefore, a highly ordered cone mosaic is advantageous for the study of retinal circuitry, since the results obtained are more reliable than with a less well ordered mosaic (Braun, Kro¨ger & Wagner, 1997). In many cichlid fishes, one short-wave sensitive (SWS) single cone is surrounded by four double cones with middle- and long-wave sensitive (MWS and LWS, respectively) members (Fernald & Liebman, 1980). The cone mosaic is of almost crystalline regularity with LWS and MWS members of double cones in alternating positions. Additionally, the relative numbers of spectrally distinct cones of 1:2:2 (SWS, MWS, LWS, respectively) are constant throughout the retina including a region of * Corresponding author. Tel.: +49-7071-2973022; fax: +49-7071294014. E-mail address: [email protected] (R.H.H. Kro¨ger)
highest cell densities in the temporal pole of the bulbus (Wagner, 1978; Fernald, 1981). It has been reported for the blue acara (Aequidens pulcher, Cichlidae) that single cones express a MWS pigment (lmax = 544 nm) while the double cones consist of identical LWS members (lmax = 617 nm) (Levine & MacNichol, 1979). Such a dichromatic organisation of the retina would further simplify the study of connectivities in the outer retina. We therefore selected the blue acara as a model system to investigate the structures participating in the initial stages of fish colour vision. In the course of our studies, however, we found that A. pulcher has three spectrally different cone types and we present here absorbance spectra measured by microspectrophotometry. Colour opponent encoding of chromatic information in cone-specific horizontal cells (CHCs) in the light adapted fish retina is due to negative feedback from CHCs to cones (Stell, Lightfoot, Wheeler & Leeper, 1975; Stell & Lightfoot, 1975; Stell, Kretz & Lightfoot, 1982; Downing & Djamgoz, 1989; Stell, Barton, Ohtsuka & Hirano, 1994; Kamermans & Spekreijse, 1995; Verweij, Kamermans & Spekreijse, 1996). We reared
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fish in monochromatic lights to interfere with the feedback loops in the outer retina by preferrential stimulation of classes of spectral cone types. This treatment induced significant changes in the morphological dynamics of the synaptic complexes between cones and second order neurons (Kro¨ger & Wagner, 1996b). In contrast, it had no or only minor effects on the patterns of CHC-cone connectivity (Braun et al., 1997). Here we present results on the absorbance spectra, morphology, and frequencies of cone photoreceptors.
2. Methods
2.1. Rearing conditions Fish were reared from 2 weeks after fertilisation of the eggs for a minimum of 1 year in 12 h light/12 h dark cycles. Monochromatic illumination was generated by passing the light of tungsten halogen bulbs through interference filters (half-maximum bandwidths: 5 – 10 nm). The spectral positions [central wavelengths 485 (blue), 534 (green), and 624 (red) nm] were chosen to match the wavelengths of maximum absorbance of blue acara cone and rod photoreceptors as reported in the literature (Levine & MacNichol, 1979). An additional group of fish was reared in monochromatic light with a central wavelength of 450 nm (violet, bandwidth: 20 nm) after we had measured cone absorbances in our fish and found that there are three instead of two spectrally different cone types. Downwelling irradiances in the centres of the empty aquaria were adjusted to 1.1 ×1012 quanta/s per cm2. Two control groups were reared in white light [downwelling illuminances of 33 lux (bright white group) and 0.2 lux (dim white group)], the latter being slightly dimmer to the human eye than the green monochromatic light (0.45 lux). In the absence of data on the spectral sensitivity of A. pulcher, photometric comparisons between light intensities appeared to be a better approximation than radiometric measurements, in particular as the human and blue acara cone complements were found to be spectrally more similar than expected. For further details on husbandry and lighting conditions see Kro¨ger and Wagner (1996a,b).
2.2. Microspectrophotometry The absorbance spectra of individual photoreceptors were measured with a modified dual-beam Liebman microspectrophotometer under computer control (Liebman & Entine, 1964; Mollon, Bowmaker & Jacobs, 1984). The measuring beam (normally about 2 mm2 cross-section) was aligned to pass transversely through a given outer segment while the reference beam passed through a clear space adjacent to the photoreceptor.
Spectra were scanned from 750 to 350 nm. A standardised computer programme was employed to estimate the wavelength of maximum absorbance (lmax) of each outer segment and only records that passed rigid selection criteria were used for subsequent detailed analysis (Mollon et al., 1984; Mansfield, 1985; Bowmaker, Astell, Hunt & Mollon, 1991).
2.3. Measurements of cone outer segment lengths Dark adapted fish were sacrificed in the dark by rapid decapitation using an infra-red viewing device (AEG). The eyes were enucleated and opened. After fixation for 2 h with 4% paraformaldehyde (PA) in 0.07% phosphate buffer, pH 7.4, the retinae were isolated. The melanin granules in the pigment epithelium were bleached with a solution of 3% H2O2 in phosphate buffered saline (PBS, pH 7.4) that was adjusted to pH 13.5 with KOH immediately before use (modified after, Strauss, 1932). Bleaching was necessary to visualise the full length of cone outer segments. After washing with PBS, the retinae were incubated in 0.2% lucifer yellow in PBS and embedded in 23% hens egg albumin and 0.5% gelatine dissolved in PBS and cured with 2.5% glutaraldehyde. 100 mm radial sections were cut on a vibratome (Oxford). Per sampling position, 16 optical sections separated by 1 mm were acquired with a confocal laser scanning microscope (ZEISS LSM 410 invert). The inner and outer segments could be followed along their full lengths in the stacks of optical sections even if the sectioning planes did not exactly parallel the longitudinal axis of the cones. The periphery of the retina and a temporal area of specialisation (Fernald, 1983) were excluded, otherwise sampling sites were chosen at random within the central portion of the retina. Due to their small size, reliable measurements of the lengths of single cone outer segments were not possible. MWS and LWS members of double cones could not be differentiated with certainty by morphological criteria. Measurements were therefore taken from all double cone members irrespective of their spectral identity. The lengths of five cone outer segments were determined per site and averaged. Three sites per retina were studied in three retinae from three fish in each group. Cone outer segment lengths (COSL) were measured relative to cone inner segment length to remove variation in absolute cone size due to differences in body size of the fish.
2.4. Quantification of cone frequencies Cone frequencies were determined in 1 mm tangential sections through the inner segment layer of light adapted retinae fixed with PA, embedded in EPON, and stained with Richardson’s solution (Richardson, Jarett & Finke, 1960). Since the observed effects were limited to a loss of single cones, unoccupied centres of
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100 adjacent double cone squares were counted for quantification, starting at a randomly chosen position in the mosaic. Three counts were performed per retinal location and averaged, and four retinae from four fish were studied in each group (data in Fig. 3e). Two retinae from two fish in each group were used to determine the dependency on eccentricity of the number of missing single cones in fish reared in monochromatic lights of short wavelengths. One count was taken per retinal location (data in Fig. 3f).
3. Results
3.1. Microspectrophotometry The cone complement of the blue acara comprises single cones and two types of double cone, each consisting of two members with their inner segments in close apposition. In identical double cones, both members are LWS with the wavelength of maximum absorbance (lmax) at about 570 nm. In the much more numerous, unequal double cones the LWS member has lmax at about 570 nm whereas the lmax of the slightly smaller MWS member is close to 530 nm. The third type of cone consists of small, SWS single cones with lmax close to 453 nm. Rods have lmax at about 500 nm (Fig. 1, Table 1). The spectral locations of these pigments and the shapes of the absorbance spectra indicate strongly that the pigments are primarily rhodopsins, but we cannot exclude the possibility that small amounts of porphyropsins are present. Ultraviolet sensitivity can be excluded in A. pulcher since the crystalline lens contains a yellow pigment with a long pass cut-off at 432 nm (Douglas & McGuigan, 1989). In general, exposure to the different monochromatic
Fig. 1. Absorbance spectra of photoreceptors measured microspectrophotometrically and the spectral positions of the monochromatic rearing lights (stippled lines). The absorbance data were obtained from the bright white group.
Fig. 2. Relative length of double cone outer segments as a function of lighting condition during development. Outer segment lengths were determined in stacks of radial optical sections of dark adapted retina and expressed as the ratio of outer segment length over inner segment length to reduce bias due to differences in body size. * PB 0.05, ** PB 0.01, *** P B0.001, Student’s t-test.
lights appears to have little or no effect on the lmax of the various spectrally distinct photoreceptor types (Table 1).
3.2. Cone outer segment lengths In the bright white group, COSLs of double cones were significantly shorter than in all other groups where illumination was dimmer (Fig. 2). In red and green light, where the SWS single cones received little or no stimulation (Fig. 1), double cone OSL was the same as in dim white light. Only in blue light did COSLs change significantly (Fig. 2): the outer segments of double cones were longer than in all other groups.
3.3. Cone frequencies Surplus cones and missing double cones were observed in negligible numbers independent of the rearing conditions. Almost all single cone positions were occupied in the bright white, dim white, red, and green groups. However, in fish reared in blue light, there was a marked reduction in the number of SWS cones with nearly 20% of the single cones missing in the central retina (Fig. 3b–e). The frequency of occupied single cone positions decreased from 100% in the periphery to about 80% in the centre of the retina (Fig. 3f). This effect was even more pronounced in fish from the violet group (Fig. 3f), suggesting a dose-dependency of the response. All single cones were present in retinae of fish reared in violet light at the ages of 4 and 8 months, whereas the full effect of elimination of single cones could be observed after 1 year of rearing in monochromatic blue and violet light. This level of single cones was stable for at least another 6 months, suggesting that the retina maturated during the first year of life. Degenerating single cones were not observed.
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Table 1 lmax of rods and cones from A. pulcher reared under different lighting conditionsa Rods
Cones SWS
Bright white Blue Green Red a
502.2 91.4; n=3 499.9 95.2; n=2 499.4; n=1 500.8; n=1
453.0 95.5; 452.3 92.2; 451.0 9 3.7; 451.3 93.5;
MWS n =3 n =5 n=2 n =4
531.2 94.2; 530.8 9 5.8; 528.8 94.4; 537.1 94.1;
LWS n = 12 n =6 n=8 n=6
571.0 9 3.5; 568.6 91.3; 568.5 9 3.8; 570.0 93.3;
n=11 n=5 n= 7 n= 15
The values given (nm) are the mean 9 S.D. of the lmax of the individual cells; n, number of cells selected for analysis.
4. Discussion
4.1. Cone absorbances The lmax for the rods and cones of the blue acara reported here are notably different from previously published data, which are themselves inconsistent (Loew & Lythgoe, 1978; Levine & MacNichol, 1979). If the expression of photopigment genes changes during development, these discrepancies could be due to differences in the age of the animals used in the different studies. However, the more likely explanation is of an incorrect classification of a morphologically similar species to A. pulcher (Loew & Lythgoe, 1978) or of tabulation errors (Levine & MacNichol, 1979). The small variations in the lmax of the MWS cones, may, if real, be due to modifications of the level of porphyropsin incorporated into the cones or by a polymorphism of the MWS cone pigment gene as has been reported in the goldfish (Johnson, Grant, Zankel, Boehm, Merbs, Nahans et al., 1993).
4.2. Double cone outer segment lengths It has been shown in rats that the photosensitivity of the retina is under regulatory control. The total number of photons absorbed during a light period remains relatively constant under different level of illumination over at least 2 orders of magnitude (photostasis). The quantum catch efficiency is adjusted by regulation of photopigment density, length of the photoreceptor outer segments, and packing of photoreceptor cells (Penn & Williams, 1986; Penn, Tolman, Thum & Koutz, 1992; Schremser & Williams, 1995a,b; Reiser, Williams & Pugh, 1996). There are also modifications in the synaptic connections of photoreceptors (Case & Plummer, 1993). The difference in COSL between the bright white and dim white groups in this study suggests that similar mechanisms are present in the fish retina. Since the shedding of cone outer segment membranes is triggered by the onset of darkness (O’Day & Young, 1978), lower modulation of light intensity between the light and dark phases might reduce the amount of material shed in the
dim white group. Such a modulation dependency would be a simple and effective mechanism to adjust photoreceptor outer segment lengths to ambient light levels and appears to be present in frogs (Gordon & Dahl, 1990). However, constant darkness can lead to both a decrease (Hollyfield & Rayborn, 1979) or an increase (Bernstein, Breding & Fisher, 1984) in photoreceptor outer segment lengths depending on the balance between membrane shedding and addition. Furthermore, if animals are switched to a different cyclic intensity, changes in both membrane removal and addition can be involved in adjusting photoreceptor lengths, depending on the experimental conditions (Schremser & Williams, 1995a). From our measurements it cannot be decided whether the increase in COSL in the dim white group is due to a reduction in membrane shedding, an increase in membrane addition, or both. Since MWS/LWS double cones are much more numerous than LWS/LWS pairs, the results of our COSL measurements are strongly biased toward unequal double cones. In the red and blue lights, total stimulation of MWS/LWS cone pairs was about the same (Fig. 1). It is therefore unlikely that the elongation of outer segments in the blue group was due to lower modulation of double cone stimulation between the light and dark phases, since in that case the same effect should have occurred in the red group. This suggests that preferential stimulation of the SWS single cones up-regulates double cone OSL. In frogs, rod disc shedding is induced by stimulation of LWS cones and it is speculated that a luminosity encoding horizontal cell is involved (Gordon & Dahl, 1990).
4.3. Loss of SWS single cones Light of short wavelength is known to be particularly harmful to retinal photoreceptors due to the high energy of its photons (Ham, Mueller & Sliney, 1976; Kirschfeld, 1982; Norren & Schellekens, 1990). In the present experiments we can exclude this type of blue light damage, since the light of the bright white group contained short wavelengths (integrated irradiance from 400 to 500 nm) at a far higher intensity (about tenfold) than the blue and violet light regimes. The loss
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Fig. 3. (a) The regular square type cone mosaic is evident in the patterns of cone outer and inner segments (COS and CIS, respectively), and perikarya (CPK) in a slightly oblique, tangential section through the light adapted retina of a blue acara reared in white light (DC, double cones; SC, single cones; RPK, rod perikarya). (b–d) In serial sections of the retina of a fish reared in blue monochromatic light, several positions in the mosaic where single cones are usually found are unoccupied (circles; b, inner segments; c, myoids; d, perikarya). All scalebars are 10 mm. (e) The number of cones missing in the central retinae was significantly higher in fish reared in blue monochromatic light than in all other groups. ** P B 0.01, *** P B0.001, Student’s t-test. (f) In the recently differentiated, peripheral retina of fish reared in blue and violet light (full and dashed lines, respectively), few single cones were missing. The transition from the complete mosaic in the periphery to the central retina occurred between 80 and 50% retinal radius.
of single cones thus occurred in response to the spectrally skewed stimulation of the cone types. Involvement of rods in this regulation of chromatic input is unlikely since, in the light adapted retina of many teleosts including the blue acara, rods are shielded from the incoming light by melanin granules of the pigment epithelium due to retinomotor movements (Douglas, 1982; Burnside & Nagle, 1983). The photoreceptor layer of the fish retina grows by expansion, i.e. enlargement of cones, and addition of new cells, mainly rod photoreceptors. Cones are added exclusively in proliferation zones near the ora serrata and the ventral fissure (Mu¨ller, 1952; Fernald, 1989;
Powers & Raymond, 1990). The decrease in single cone frequency from the periphery toward the centre of the retina in the blue and violet groups (Fig. 3f) indicates that single cones are formed at a normal rate in the growing retina and selectively eliminated later in development. Since degenerating cones could not be observed, this elimination appears to be rapid. A similar pattern of retinal development is found in salmonids where ultra-violet sensitive (UVS) cones are formed in the proliferation zones at all ages, but are short lived in older fish (Kunz, Wildenburg, Goodrich & Callaghan, 1994). Thyroid hormones appear to be involved in the maintenance and elimination of salmonid UV photo-
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sensitivity (Browman & Hawryshyn, 1994), suggesting a systemic, global control of cone survival. Hormonal control is also suggested by the coincidence of sexual maturation and the loss of single cones between 8 and 12 months of age. However, retinal mechanisms could also be responsible for the elimination of SWS cones in A. pulcher reared in blue light. Feedforward and feedback synapses with CHCs (Stell & Lightfoot, 1975; Downing & Djamgoz, 1989Stell et al., 1994; Kamermans & Spekreijse, 1995) are a possible pathway for signal transmission.
4.4. General discussion The reduction of the frequency of SWS cones and the elongation of the outer segments of MWS and LWS cones in fish reared in light of short wavelengths will both shift the spectral sensitivity of the retina from short to long wavelengths, thus counteracting the preferential stimulation in the short wavelength band. The SWS single cones appear to play a key role in this regulation, since only blue light, but not red light, induced such changes. Previous experiments involving long term exposure or rearing in monochromatic light regimes, scotopic illumination, and darkness apparently had little or no effect on colour vision related behaviour in primates (Boothe, Teller & Sackett, 1975; Brenner, Schelvis & Nuboer, 1985; Di, Neitz & Jacobs, 1987; Brenner, Cornelissen & Nuboer, 1990), tree shrews (Petry & Kelly, 1991), and pigeons (Brenner, Spaan, Wortel & Nuboer, 1983). Ground squirrels growing up in darkness or red light reached the adult chromatic organisation of fibers in the optic nerve, albeit the development was delayed (McCourt & Jacobs, 1983). Ducks reared in the light of a sodium lamp (589 nm) learned to discriminate between wavelengths, although initially the wavelength of the stimulus exerted no control over their responses (Peterson, 1961). In goldfish, monochromatic red light had only small, statistically non-significant behavioural effects; rearing in blue monochromatic light induced no change at all (Mecke, 1983). Effects of spectral deprivation thus appear to be minor or absent on higher levels of chromatic processing and in the behavioural responses. Our findings in the initial stages of the chromatic pathways and on the cellular and subcellular levels, on the other hand, demonstrate that specific changes do occur and suggest that the retina of fish responds to abnormal spectral composition of the visual environment in a compensatory fashion. This apparent difference between the cellular and systemic levels in the consequences of spectral deprivation may be due to the organisation of the visual system which is designed to process contrasts rather than absolute changes in brightness. This appears to favour compensatory mech-
anisms which make it difficult to detect effects of chromatic deprivation on colour vision in systemic, behavioural tests. For instance, the reduction of the number of SWS cones by a relatively small amount might not be detectable from spectral sensitivity functions unless very careful increment threshold type experiments were performed. Furthermore, changes in the relative number of cones would have only minor effects on wavelength discrimination, since input from all spectral cone types would still be available. The loss of single cones, however, is a lasting effect and cannot be reversed within a short time of testing. Fishes reared in monochromatic lights therefore are good models to study the compensatory mechanisms of the visual system and their locations. We are currently investigating the effects on second order neurons, i.e. horizontal cells. An H1-CHC loses more than 50% of its short-wave input if the single cone in the centre of its dendritic field is missing (Braun et al., 1997) and this should be detectable in the spectral response characteristics of the cells. Innate behaviours like the optomotor and dorsal light responses, which do not require training of the animals under lighting conditions different from the illumination during rearing, can be used to investigate spectral sensitivity on the systemic level.
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McCourt, M. E., & Jacobs, G. H. (1983). Effects of photic environment on the development of spectral response properties of optic nerve fibers in the ground squirrel. Experimental Brain Research, 49, 443 – 452. Mecke, E. (1983). Absence of changes in colour discrimination ability of goldfish when reared in monochromatic light. Annales Zoologica Fennici, 20, 239 – 244. Mollon, J. D., Bowmaker, J. K., & Jacobs, G. H. (1984). Variations of colour vision in a new world primate can be explained by polymorphism of retinal photopigments. Proceedings of the Royal Society of London B, 222, 373 – 399. Mu¨ller, H. (1952). Bau und Wachstum der Netzhaut des Guppy (Lebistes reticulatus). Zoologische Jahrbu¨cher (Allgemeine Zoologie und Physiologie der Tiere), 63, 275 – 324. Norren, D. V., & Schellekens, P. (1990). Blue light hazard in rat. Vision Research, 30, 1517 – 1520. O’Day, W. T., & Young, R. W. (1978). Rhythmic daily shedding of outer-segment membranes by visual cells in the goldfish. Journal of Cell Biology, 76, 593 – 604. Penn, J. S., Tolman, B. L., Thum, L. A., & Koutz, C. A. (1992). Effect of light history on the rat retina: timecourse of morphological adaptation and readaptation. Neurochemical Research, 17, 91 – 99. Penn, J. S., & Williams, T. D. (1986). Photostasis: regulation of daily photon-catch by rat retinas in response to various cyclic illuminances. Experimental Eye Research, 43, 915 – 928. Peterson, N. (1961). Effects of monochromatic rearing on the control of responding by wavelength. Science, 136, 774 – 775. Petry, H. M., & Kelly, J. P. (1991). Psychophysical measurement of spectral sensitivity and color vision in red-light-reared tree shrews (Tupaia belangeri ). Vision Research, 31, 1749 – 1757. Powers, M. K., & Raymond, P. A. (1990). Development of the visual system. In R. H. Douglas, & M. B. A. Djamgoz, The 6isual system of fish (pp. 419 – 442). London: Chapman and Hall. Reiser, M. A., Williams, T. P., & Pugh, E. N. (1996). The effect of light history on the aspartate-isolated fast PIII responses of the albino rat retina. In6estigati6e Ophthalmology and Visual Science, 37, 221 – 229. Richardson, K. C., Jarett, L., & Finke, E. H. (1960). Embedding in Epoxy resins for ultrathin sectioning in electron microscopy. Stain Technology, 35, 313 – 323. Schremser, J. L., & Williams, T. P. (1995). Rod outer segment (ros) renewal as a mechanism for adaptation to a new intensity environment. 1. rhodopsin levels and ros length. Experimental Eye Research, 61, 17 – 23. Schremser, J. L., & Williams, T. P. (1995). Rod outer segment (ros) renewal as a mechanism for adaptation to a new intensity environment. 2. rhodopsin synthesis and packing density. Experimental Eye Research, 61, 25 – 32. Stell, W. K., Lightfoot, D. O., Wheeler, T., & Leeper, H. (1975). Goldfish retina: functional polarization of cone horizontal cell dendrites and synapses. Science, 190, 989 – 990. Stell, W. K., & Lightfoot, D. O. (1975). Color specific interconnection of cones and horizontal cells in the retina of the goldfish. Journal of Comparati6e Neurology, 159, 473 – 502. Stell, W. K., Kretz, R., & Lightfoot, D. O. (1982). Horizontal cell connectivity in goldfish. In B. D. Drujan, & M. Laufer, The S-Potential (pp. 51 – 75). New York: Alan Liss. Stell, W. K., Barton, L., Ohtsuka, T., & Hirano, J. (1994). Chromatic and neurochemical correlates of synapses between cones and horizontal cells. In R. J. Olson, & E. M. Lasater, Great basin 6isual science symposium (pp. 41 – 48). Salt Lake City: University of Utah. Strauss, A. (1932). U8 ber die Bleichung des Melanins. Zeitschrift fu¨r wissenschaftliche Mikroskopie, 49, 123 – 125.
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Morphological changes in the retina of Aequidens pulcher (Cichlidae) after rearing in monochromatic light R.H.H. Kro¨ger a,*, J.K. Bowmaker b, H.J. Wagner a a
Anatomisches Institut, Eberhard-Karls-Uni6ersita¨t Tu¨bingen, O8 sterbergstraße 3, 72074 Tu¨bingen, Germany b Institute of Ophthalmology, Uni6ersity College London, Bath Street, London EC1V 9EL, UK Received 8 May 1998; received in revised form 23 July 1998
Abstract We investigate the processing of chromatic information in the outer retina of a cichlid fish, Aequidens pulcher. The colour opponent response characteristics of some classes of cone-specific horizontal cells in the fish retina are the result of feedforward– feedback loops with cone photoreceptors. To interfere with the reciprocal transmissions of signals, animals were reared in monochromatic lights which preferentially stimulated the spectrally different cone types. Here we report the effects on the cones. Their absorbance spectra were largely unaffected, indicating no change in photopigment gene expression. Significant changes were observed in the cone outer segment lengths and the frequencies of spectral cone types. Quantum catch efficiency and survival of cones appear to be controlled in a spectrally selective way. Our results suggest that the retina responds to spectral deprivation in a compensatory fashion aimed at balancing the input from the different cone types to second order neurons. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Fish; Color vision; Photopigment gene expression; Photoreceptor survival; Adaptive plasticity
1. Introduction Investigations of chromatic information processing in the retina and its cellular substrate are critically dependent on unambiguous identification of spectral cone types (Stell, Barton, Ohtsuka & Hirano, 1994). Therefore, a highly ordered cone mosaic is advantageous for the study of retinal circuitry, since the results obtained are more reliable than with a less well ordered mosaic (Braun, Kro¨ger & Wagner, 1997). In many cichlid fishes, one short-wave sensitive (SWS) single cone is surrounded by four double cones with middle- and long-wave sensitive (MWS and LWS, respectively) members (Fernald & Liebman, 1980). The cone mosaic is of almost crystalline regularity with LWS and MWS members of double cones in alternating positions. Additionally, the relative numbers of spectrally distinct cones of 1:2:2 (SWS, MWS, LWS, respectively) are constant throughout the retina including a region of * Corresponding author. Tel.: +49-7071-2973022; fax: +49-7071294014. E-mail address: [email protected] (R.H.H. Kro¨ger)
highest cell densities in the temporal pole of the bulbus (Wagner, 1978; Fernald, 1981). It has been reported for the blue acara (Aequidens pulcher, Cichlidae) that single cones express a MWS pigment (lmax = 544 nm) while the double cones consist of identical LWS members (lmax = 617 nm) (Levine & MacNichol, 1979). Such a dichromatic organisation of the retina would further simplify the study of connectivities in the outer retina. We therefore selected the blue acara as a model system to investigate the structures participating in the initial stages of fish colour vision. In the course of our studies, however, we found that A. pulcher has three spectrally different cone types and we present here absorbance spectra measured by microspectrophotometry. Colour opponent encoding of chromatic information in cone-specific horizontal cells (CHCs) in the light adapted fish retina is due to negative feedback from CHCs to cones (Stell, Lightfoot, Wheeler & Leeper, 1975; Stell & Lightfoot, 1975; Stell, Kretz & Lightfoot, 1982; Downing & Djamgoz, 1989; Stell, Barton, Ohtsuka & Hirano, 1994; Kamermans & Spekreijse, 1995; Verweij, Kamermans & Spekreijse, 1996). We reared
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fish in monochromatic lights to interfere with the feedback loops in the outer retina by preferrential stimulation of classes of spectral cone types. This treatment induced significant changes in the morphological dynamics of the synaptic complexes between cones and second order neurons (Kro¨ger & Wagner, 1996b). In contrast, it had no or only minor effects on the patterns of CHC-cone connectivity (Braun et al., 1997). Here we present results on the absorbance spectra, morphology, and frequencies of cone photoreceptors.
2. Methods
2.1. Rearing conditions Fish were reared from 2 weeks after fertilisation of the eggs for a minimum of 1 year in 12 h light/12 h dark cycles. Monochromatic illumination was generated by passing the light of tungsten halogen bulbs through interference filters (half-maximum bandwidths: 5 – 10 nm). The spectral positions [central wavelengths 485 (blue), 534 (green), and 624 (red) nm] were chosen to match the wavelengths of maximum absorbance of blue acara cone and rod photoreceptors as reported in the literature (Levine & MacNichol, 1979). An additional group of fish was reared in monochromatic light with a central wavelength of 450 nm (violet, bandwidth: 20 nm) after we had measured cone absorbances in our fish and found that there are three instead of two spectrally different cone types. Downwelling irradiances in the centres of the empty aquaria were adjusted to 1.1 ×1012 quanta/s per cm2. Two control groups were reared in white light [downwelling illuminances of 33 lux (bright white group) and 0.2 lux (dim white group)], the latter being slightly dimmer to the human eye than the green monochromatic light (0.45 lux). In the absence of data on the spectral sensitivity of A. pulcher, photometric comparisons between light intensities appeared to be a better approximation than radiometric measurements, in particular as the human and blue acara cone complements were found to be spectrally more similar than expected. For further details on husbandry and lighting conditions see Kro¨ger and Wagner (1996a,b).
2.2. Microspectrophotometry The absorbance spectra of individual photoreceptors were measured with a modified dual-beam Liebman microspectrophotometer under computer control (Liebman & Entine, 1964; Mollon, Bowmaker & Jacobs, 1984). The measuring beam (normally about 2 mm2 cross-section) was aligned to pass transversely through a given outer segment while the reference beam passed through a clear space adjacent to the photoreceptor.
Spectra were scanned from 750 to 350 nm. A standardised computer programme was employed to estimate the wavelength of maximum absorbance (lmax) of each outer segment and only records that passed rigid selection criteria were used for subsequent detailed analysis (Mollon et al., 1984; Mansfield, 1985; Bowmaker, Astell, Hunt & Mollon, 1991).
2.3. Measurements of cone outer segment lengths Dark adapted fish were sacrificed in the dark by rapid decapitation using an infra-red viewing device (AEG). The eyes were enucleated and opened. After fixation for 2 h with 4% paraformaldehyde (PA) in 0.07% phosphate buffer, pH 7.4, the retinae were isolated. The melanin granules in the pigment epithelium were bleached with a solution of 3% H2O2 in phosphate buffered saline (PBS, pH 7.4) that was adjusted to pH 13.5 with KOH immediately before use (modified after, Strauss, 1932). Bleaching was necessary to visualise the full length of cone outer segments. After washing with PBS, the retinae were incubated in 0.2% lucifer yellow in PBS and embedded in 23% hens egg albumin and 0.5% gelatine dissolved in PBS and cured with 2.5% glutaraldehyde. 100 mm radial sections were cut on a vibratome (Oxford). Per sampling position, 16 optical sections separated by 1 mm were acquired with a confocal laser scanning microscope (ZEISS LSM 410 invert). The inner and outer segments could be followed along their full lengths in the stacks of optical sections even if the sectioning planes did not exactly parallel the longitudinal axis of the cones. The periphery of the retina and a temporal area of specialisation (Fernald, 1983) were excluded, otherwise sampling sites were chosen at random within the central portion of the retina. Due to their small size, reliable measurements of the lengths of single cone outer segments were not possible. MWS and LWS members of double cones could not be differentiated with certainty by morphological criteria. Measurements were therefore taken from all double cone members irrespective of their spectral identity. The lengths of five cone outer segments were determined per site and averaged. Three sites per retina were studied in three retinae from three fish in each group. Cone outer segment lengths (COSL) were measured relative to cone inner segment length to remove variation in absolute cone size due to differences in body size of the fish.
2.4. Quantification of cone frequencies Cone frequencies were determined in 1 mm tangential sections through the inner segment layer of light adapted retinae fixed with PA, embedded in EPON, and stained with Richardson’s solution (Richardson, Jarett & Finke, 1960). Since the observed effects were limited to a loss of single cones, unoccupied centres of
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100 adjacent double cone squares were counted for quantification, starting at a randomly chosen position in the mosaic. Three counts were performed per retinal location and averaged, and four retinae from four fish were studied in each group (data in Fig. 3e). Two retinae from two fish in each group were used to determine the dependency on eccentricity of the number of missing single cones in fish reared in monochromatic lights of short wavelengths. One count was taken per retinal location (data in Fig. 3f).
3. Results
3.1. Microspectrophotometry The cone complement of the blue acara comprises single cones and two types of double cone, each consisting of two members with their inner segments in close apposition. In identical double cones, both members are LWS with the wavelength of maximum absorbance (lmax) at about 570 nm. In the much more numerous, unequal double cones the LWS member has lmax at about 570 nm whereas the lmax of the slightly smaller MWS member is close to 530 nm. The third type of cone consists of small, SWS single cones with lmax close to 453 nm. Rods have lmax at about 500 nm (Fig. 1, Table 1). The spectral locations of these pigments and the shapes of the absorbance spectra indicate strongly that the pigments are primarily rhodopsins, but we cannot exclude the possibility that small amounts of porphyropsins are present. Ultraviolet sensitivity can be excluded in A. pulcher since the crystalline lens contains a yellow pigment with a long pass cut-off at 432 nm (Douglas & McGuigan, 1989). In general, exposure to the different monochromatic
Fig. 1. Absorbance spectra of photoreceptors measured microspectrophotometrically and the spectral positions of the monochromatic rearing lights (stippled lines). The absorbance data were obtained from the bright white group.
Fig. 2. Relative length of double cone outer segments as a function of lighting condition during development. Outer segment lengths were determined in stacks of radial optical sections of dark adapted retina and expressed as the ratio of outer segment length over inner segment length to reduce bias due to differences in body size. * PB 0.05, ** PB 0.01, *** P B0.001, Student’s t-test.
lights appears to have little or no effect on the lmax of the various spectrally distinct photoreceptor types (Table 1).
3.2. Cone outer segment lengths In the bright white group, COSLs of double cones were significantly shorter than in all other groups where illumination was dimmer (Fig. 2). In red and green light, where the SWS single cones received little or no stimulation (Fig. 1), double cone OSL was the same as in dim white light. Only in blue light did COSLs change significantly (Fig. 2): the outer segments of double cones were longer than in all other groups.
3.3. Cone frequencies Surplus cones and missing double cones were observed in negligible numbers independent of the rearing conditions. Almost all single cone positions were occupied in the bright white, dim white, red, and green groups. However, in fish reared in blue light, there was a marked reduction in the number of SWS cones with nearly 20% of the single cones missing in the central retina (Fig. 3b–e). The frequency of occupied single cone positions decreased from 100% in the periphery to about 80% in the centre of the retina (Fig. 3f). This effect was even more pronounced in fish from the violet group (Fig. 3f), suggesting a dose-dependency of the response. All single cones were present in retinae of fish reared in violet light at the ages of 4 and 8 months, whereas the full effect of elimination of single cones could be observed after 1 year of rearing in monochromatic blue and violet light. This level of single cones was stable for at least another 6 months, suggesting that the retina maturated during the first year of life. Degenerating single cones were not observed.
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Table 1 lmax of rods and cones from A. pulcher reared under different lighting conditionsa Rods
Cones SWS
Bright white Blue Green Red a
502.2 91.4; n=3 499.9 95.2; n=2 499.4; n=1 500.8; n=1
453.0 95.5; 452.3 92.2; 451.0 9 3.7; 451.3 93.5;
MWS n =3 n =5 n=2 n =4
531.2 94.2; 530.8 9 5.8; 528.8 94.4; 537.1 94.1;
LWS n = 12 n =6 n=8 n=6
571.0 9 3.5; 568.6 91.3; 568.5 9 3.8; 570.0 93.3;
n=11 n=5 n= 7 n= 15
The values given (nm) are the mean 9 S.D. of the lmax of the individual cells; n, number of cells selected for analysis.
4. Discussion
4.1. Cone absorbances The lmax for the rods and cones of the blue acara reported here are notably different from previously published data, which are themselves inconsistent (Loew & Lythgoe, 1978; Levine & MacNichol, 1979). If the expression of photopigment genes changes during development, these discrepancies could be due to differences in the age of the animals used in the different studies. However, the more likely explanation is of an incorrect classification of a morphologically similar species to A. pulcher (Loew & Lythgoe, 1978) or of tabulation errors (Levine & MacNichol, 1979). The small variations in the lmax of the MWS cones, may, if real, be due to modifications of the level of porphyropsin incorporated into the cones or by a polymorphism of the MWS cone pigment gene as has been reported in the goldfish (Johnson, Grant, Zankel, Boehm, Merbs, Nahans et al., 1993).
4.2. Double cone outer segment lengths It has been shown in rats that the photosensitivity of the retina is under regulatory control. The total number of photons absorbed during a light period remains relatively constant under different level of illumination over at least 2 orders of magnitude (photostasis). The quantum catch efficiency is adjusted by regulation of photopigment density, length of the photoreceptor outer segments, and packing of photoreceptor cells (Penn & Williams, 1986; Penn, Tolman, Thum & Koutz, 1992; Schremser & Williams, 1995a,b; Reiser, Williams & Pugh, 1996). There are also modifications in the synaptic connections of photoreceptors (Case & Plummer, 1993). The difference in COSL between the bright white and dim white groups in this study suggests that similar mechanisms are present in the fish retina. Since the shedding of cone outer segment membranes is triggered by the onset of darkness (O’Day & Young, 1978), lower modulation of light intensity between the light and dark phases might reduce the amount of material shed in the
dim white group. Such a modulation dependency would be a simple and effective mechanism to adjust photoreceptor outer segment lengths to ambient light levels and appears to be present in frogs (Gordon & Dahl, 1990). However, constant darkness can lead to both a decrease (Hollyfield & Rayborn, 1979) or an increase (Bernstein, Breding & Fisher, 1984) in photoreceptor outer segment lengths depending on the balance between membrane shedding and addition. Furthermore, if animals are switched to a different cyclic intensity, changes in both membrane removal and addition can be involved in adjusting photoreceptor lengths, depending on the experimental conditions (Schremser & Williams, 1995a). From our measurements it cannot be decided whether the increase in COSL in the dim white group is due to a reduction in membrane shedding, an increase in membrane addition, or both. Since MWS/LWS double cones are much more numerous than LWS/LWS pairs, the results of our COSL measurements are strongly biased toward unequal double cones. In the red and blue lights, total stimulation of MWS/LWS cone pairs was about the same (Fig. 1). It is therefore unlikely that the elongation of outer segments in the blue group was due to lower modulation of double cone stimulation between the light and dark phases, since in that case the same effect should have occurred in the red group. This suggests that preferential stimulation of the SWS single cones up-regulates double cone OSL. In frogs, rod disc shedding is induced by stimulation of LWS cones and it is speculated that a luminosity encoding horizontal cell is involved (Gordon & Dahl, 1990).
4.3. Loss of SWS single cones Light of short wavelength is known to be particularly harmful to retinal photoreceptors due to the high energy of its photons (Ham, Mueller & Sliney, 1976; Kirschfeld, 1982; Norren & Schellekens, 1990). In the present experiments we can exclude this type of blue light damage, since the light of the bright white group contained short wavelengths (integrated irradiance from 400 to 500 nm) at a far higher intensity (about tenfold) than the blue and violet light regimes. The loss
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Fig. 3. (a) The regular square type cone mosaic is evident in the patterns of cone outer and inner segments (COS and CIS, respectively), and perikarya (CPK) in a slightly oblique, tangential section through the light adapted retina of a blue acara reared in white light (DC, double cones; SC, single cones; RPK, rod perikarya). (b–d) In serial sections of the retina of a fish reared in blue monochromatic light, several positions in the mosaic where single cones are usually found are unoccupied (circles; b, inner segments; c, myoids; d, perikarya). All scalebars are 10 mm. (e) The number of cones missing in the central retinae was significantly higher in fish reared in blue monochromatic light than in all other groups. ** P B 0.01, *** P B0.001, Student’s t-test. (f) In the recently differentiated, peripheral retina of fish reared in blue and violet light (full and dashed lines, respectively), few single cones were missing. The transition from the complete mosaic in the periphery to the central retina occurred between 80 and 50% retinal radius.
of single cones thus occurred in response to the spectrally skewed stimulation of the cone types. Involvement of rods in this regulation of chromatic input is unlikely since, in the light adapted retina of many teleosts including the blue acara, rods are shielded from the incoming light by melanin granules of the pigment epithelium due to retinomotor movements (Douglas, 1982; Burnside & Nagle, 1983). The photoreceptor layer of the fish retina grows by expansion, i.e. enlargement of cones, and addition of new cells, mainly rod photoreceptors. Cones are added exclusively in proliferation zones near the ora serrata and the ventral fissure (Mu¨ller, 1952; Fernald, 1989;
Powers & Raymond, 1990). The decrease in single cone frequency from the periphery toward the centre of the retina in the blue and violet groups (Fig. 3f) indicates that single cones are formed at a normal rate in the growing retina and selectively eliminated later in development. Since degenerating cones could not be observed, this elimination appears to be rapid. A similar pattern of retinal development is found in salmonids where ultra-violet sensitive (UVS) cones are formed in the proliferation zones at all ages, but are short lived in older fish (Kunz, Wildenburg, Goodrich & Callaghan, 1994). Thyroid hormones appear to be involved in the maintenance and elimination of salmonid UV photo-
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sensitivity (Browman & Hawryshyn, 1994), suggesting a systemic, global control of cone survival. Hormonal control is also suggested by the coincidence of sexual maturation and the loss of single cones between 8 and 12 months of age. However, retinal mechanisms could also be responsible for the elimination of SWS cones in A. pulcher reared in blue light. Feedforward and feedback synapses with CHCs (Stell & Lightfoot, 1975; Downing & Djamgoz, 1989Stell et al., 1994; Kamermans & Spekreijse, 1995) are a possible pathway for signal transmission.
4.4. General discussion The reduction of the frequency of SWS cones and the elongation of the outer segments of MWS and LWS cones in fish reared in light of short wavelengths will both shift the spectral sensitivity of the retina from short to long wavelengths, thus counteracting the preferential stimulation in the short wavelength band. The SWS single cones appear to play a key role in this regulation, since only blue light, but not red light, induced such changes. Previous experiments involving long term exposure or rearing in monochromatic light regimes, scotopic illumination, and darkness apparently had little or no effect on colour vision related behaviour in primates (Boothe, Teller & Sackett, 1975; Brenner, Schelvis & Nuboer, 1985; Di, Neitz & Jacobs, 1987; Brenner, Cornelissen & Nuboer, 1990), tree shrews (Petry & Kelly, 1991), and pigeons (Brenner, Spaan, Wortel & Nuboer, 1983). Ground squirrels growing up in darkness or red light reached the adult chromatic organisation of fibers in the optic nerve, albeit the development was delayed (McCourt & Jacobs, 1983). Ducks reared in the light of a sodium lamp (589 nm) learned to discriminate between wavelengths, although initially the wavelength of the stimulus exerted no control over their responses (Peterson, 1961). In goldfish, monochromatic red light had only small, statistically non-significant behavioural effects; rearing in blue monochromatic light induced no change at all (Mecke, 1983). Effects of spectral deprivation thus appear to be minor or absent on higher levels of chromatic processing and in the behavioural responses. Our findings in the initial stages of the chromatic pathways and on the cellular and subcellular levels, on the other hand, demonstrate that specific changes do occur and suggest that the retina of fish responds to abnormal spectral composition of the visual environment in a compensatory fashion. This apparent difference between the cellular and systemic levels in the consequences of spectral deprivation may be due to the organisation of the visual system which is designed to process contrasts rather than absolute changes in brightness. This appears to favour compensatory mech-
anisms which make it difficult to detect effects of chromatic deprivation on colour vision in systemic, behavioural tests. For instance, the reduction of the number of SWS cones by a relatively small amount might not be detectable from spectral sensitivity functions unless very careful increment threshold type experiments were performed. Furthermore, changes in the relative number of cones would have only minor effects on wavelength discrimination, since input from all spectral cone types would still be available. The loss of single cones, however, is a lasting effect and cannot be reversed within a short time of testing. Fishes reared in monochromatic lights therefore are good models to study the compensatory mechanisms of the visual system and their locations. We are currently investigating the effects on second order neurons, i.e. horizontal cells. An H1-CHC loses more than 50% of its short-wave input if the single cone in the centre of its dendritic field is missing (Braun et al., 1997) and this should be detectable in the spectral response characteristics of the cells. Innate behaviours like the optomotor and dorsal light responses, which do not require training of the animals under lighting conditions different from the illumination during rearing, can be used to investigate spectral sensitivity on the systemic level.
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